Antibodies can recognize and target almost any molecule with high specificity and affinity. This characteristic has been exploited to turn these natural proteins into powerful tools for diagnostic and therapeutic applications. Advances in recombinant DNA technology have facilitated the manipulation, cloning, and expression of antibody genes in a wide variety of non-lymphoid cells (Skerra, 1988; Martineau, 1998; Verma, 1998). A number of different antibody fragments have been constructed to best suit the various applications. The smallest entity that retains the full antigen-binding capacity of the whole parental immunoglobulin is the single-chain Fv fragment (scFv) (Bird, 1988). This antibody fragment comprises the variable regions of the heavy and the light chains linked by a flexible peptide-linker, which allows the expression of the protein from a single gene.
Antibody fragments have several important advantages in comparison to the entire immunoglobulin molecule. Due to their smaller size, the expression is facilitated and the yield is enhanced in a variety of expression host cells, such as E. coli cells (Plückthun, 1996). Moreover, antibody fragments allow improved tumour penetration in in vivo applications (Yokota, 1992) and they can be linked covalently to various effector molecules for therapeutic approaches.
Naturally occurring antibodies, which are secreted by plasma cells, have evolved to function in an extracellular, oxidizing environment. To obtain their functional, folded structure, they generally require the formation of disulfide-bridges within the separate domains, which are crucial for the stability of the immunoglobulin fold. In contrast to full-length antibodies, scFv or Fab antibody fragments can, in principle, be functionally expressed in a reducing environment inside any cell and directed to any compartment to target intracellular proteins and thus evoke specific biological effects (Biocca, 1991). Indeed, some intracellular single chain antibody fragments, which are called intrabodies, have been applied successfully to modulate the function of intracellular target proteins in different biological systems. Thus, resistance against viral infections has been demonstrated in plant biotechnology (Tavladoraki, 1993; Benvenuto, 1995), binding of intrabodies to HIV proteins has been shown (Rondon, 1997), and binding to oncogene products (Biocca, 1993; Cochet, 1998; Lener, 2000) has been described. Moreover, intracellular antibodies promise to be a valuable tool in characterizing the function of a vast number of genes now identified through the sequencing of the human genome (Richardson, 1995; Marasco, 1997). For example, they can be used in a functional genomics approach to block or modulate the activity of newly identified proteins, thereby contributing to the understanding of their functions. Finally, intrabodies have potential diagnostic and therapeutic applications, for example in gene therapy settings.
Despite these great prospects, the generation of functional intrabodies is still limited by their instability and insolubility or propensity to aggregate. The reducing environment of the cytoplasm prevents the formation of the conserved intrachain disulfide bridges, thus rendering a high percentage of antibody fragments unstable and, as a consequence, non-functional inside the cell (Biocca, 1995; Proba, 1997). Stability and solubility of antibody fragments therefore represents a major obstacle for the application of intrabodies as potential modulators of protein function in vivo. So far, no predictions can be made about the sequence requirements that render an antibody fragment functional in an intracellular environment.
There is, therefore, a need for antibody fragments which perform well in a broad range of different cell types and can thus be used as frameworks for diverse binding specificities. Such frameworks can be used to construct libraries for intracellular screening or can serve as an acceptor for the binding portions of an existing antibody.
Besides being uniquely suited for intracellular applications, such antibody fragments or whole antibodies based on very stable variable domain frameworks also have a distinct advantage over other antibodies in numerous extracellular and in vitro applications. When such frameworks are produced in an oxidizing environment, their disulfide-bridges can be formed, further enhancing their stability and making them highly resistant towards aggregation and protease degradation. The in vivo half-life (and thus the resistance towards aggregation and degradation by serum proteases) is, besides affinity and specificity, the single-most important factor for the success of antibodies in therapeutic or diagnostic applications (Willuda, 1999). The half-life of antibody fragments can further be increased through the covalent attachment of polymer molecules such as poly-ethylene glycol (PEG) (Weir, 2002). Stable molecules of this type represent a significant advance in the use of antibodies, especially, but not exclusively, when the Fc functionality is not desired.
The great practical importance of antibody-fragment libraries has motivated research in this area. Winter (EP 0368684) has provided the initial cloning and expression of antibody variable region genes. Starting from these genes he has created large antibody libraries having high diversity in both the complementary determining regions (CDRs) as well as in the framework regions. Winter does not disclose, however, the usefulness of different frameworks for library construction.
The teaching of Plückthun (EP 0859841), on the other hand, has tried to improve the library design by limiting the frameworks to a defined number of synthetic consensus sequences. Protein engineering efforts involving introduction of a large amount of rationally designed mutations have previously suggested mutations towards the respective consensus sequence as a suitable means for the improvement of the stability of isolated variable immunoglobulin domains (Ohage 1999; Ohage 1999 and U.S. Pat. No. 5,854,027, hereby incorporated by reference).
Plückthun (EP 0859841) discloses methods for the further optimization of binding affinities based on these consensus sequences. The Plückthun patent also acknowledges the ongoing increase in knowledge concerning antibodies and accordingly aims at including such future findings in the library design. However, no possible further improvements of the synthetic consensus frameworks are suggested.
The teachings of Winter, Plückthun and others (e.g. Soderlind, WO 0175091) have thus tried to create large antibody libraries with a focus on high diversity in the CDRs for selection and application of the selected scFvs under oxidizing conditions. All of these libraries are, however, not optimized for intracellular applications and thus not useful for selection and applications in a reducing environment, or other conditions which set special requirements on stability and solubility of the expressed antibody fragment.
The qualities required for antibody fragments to perform well in a reducing environment, e.g. the cytoplasm of prokaryotic and eukaryotic cells, are not clear. The application of intracellular antibodies or “intrabodies” is therefore currently limited by their unpredictable behavior under reducing conditions, which can affect their stability and solubility properties (Biocca, 1995; Wörn, 2000). Present patent applications (EP1040201, EP1166121 and WO0200729) and publications (Visintin, 1999) concerning intracellular screening for intrabodies focus on the screening technology but do not disclose specific antibody sequences which are functional in eukaryotic cells, in particular in yeast, and, thus, useful for library construction in this context.
Visintin and Tse have independently described the isolation of a so-called intracellular consensus sequence (ICS) (Visintin, 2002; Tse, 2002). This sequence was derived from a number of sequences that had been isolated from an antigen-antibody-interaction screen in yeast. The input into the intracellular screen was, however, heavily biased due to prior phage-display selection. Thus, all but one of the input-sequences belonged to the VH 3 subgroup in the case of Visintin et al. The published consensus sequence ICS is fully identical to the consensus sequence for the human VH 3 subgroup described by Knappik (2000) and EP0859841. VH1a consensus sequence
(Seq. Id. No. 14)QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGG IIPIFGTAANYAQKFQGRVTITADESTSTAYMELSSLRSEADTAVYYCAR WGGDGFYAMDYWGQGTLVTVSSand VH1b consensus sequence
(Seq Id. No. 15)QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGW INPNSGGTBNYAQKFQGRVTMTRDTSISTAYMELSSLRSEBDTAVYYCAR WGGDGFYAMDYWGQGTLVTVSS60 of the 62 amino acids of the ICS are also identical to the general human VH-domain consensus sequence which was proposed by Steipe as a basis for the construction of variable domains with enhanced stability (U.S. Pat. No. 6,262,238, hereby incorporated by reference). These works were, in turn, based on earlier sequence collections (i.e., Kabat, 1991 and definitions of variable domain subgroups and structural determinants (Tomlinson, 1992; Williams, 1996; Chothia, 1989 and Chothia, 1987). However, because the input to the intrabody selection was so heavily biased (i.e., in the case of Visintin et al. all but one of the VH domains was VH3), the isolation of VH3 sequences from intracellular screening is not particularly surprising. Due to the heavy bias of their input library, the work of Tse et al. and Visintin et al. does not provide a thorough evaluation of the human variable domain repertoire as would be provided by an unbiased inquiry and as is required to identify the useful intrabody frameworks present in the human repertoire.
We have previously described a system, which allows for the selection of stable and soluble intrabodies in yeast, independent of their antigen-binding specificity (Auf der Maur (2001), WO0148017). This approach allows efficient screening of scFv libraries and the isolation of specific frameworks, which are stable and soluble in the reducing environment of the yeast cell. The objective remains to actually isolate framework sequences and use the patterns in a first step to predict what sequence types would be most stable in the reducing environment and in a second step identify by analysis, recombination and further in vivo and in vitro experiments the optimal sequence.